title: Further characterisation of rotavirus cores: Ss(+)RNAs can be packaged in vitro but packaging lacks sequence specificity.
abstract: Rotavirus (RV) cores were released from double-layered particles (DLPs) by high concentrations of CaCl2, purified and 'opened' by treatment with EDTA or EGTA. Under appropriate in vitro conditions DLPs have been shown to have transcriptase and 'open cores' replicase activity. Furthermore, it has been demonstrated that transcriptase activity and infectivity of native cores can be restored by transcapsidation with VP6, VP7 and VP4. The missing link for particle reconstitution in vitro has been the manipulation of 'open cores' to become functionally active cores again. The experiments described here were undertaken with the aim of exploring packaging of RV RNAs into opened cores in vitro. Rotavirus cores were opened by approximately 200μM EGTA, leading to the release of genomic dsRNA. Conversely, RV cores were found to be stable in the presence of minimum concentrations of Ca(2+), Mg(2+), spermidine(3+) and cobalthexamine(3+) of between 40 and 300 μM. Aggregates of purified cores were resolved in the presence of 0.3mM deoxycholate (minimum concentration). Core shells opened with EGTA were reconstituted by the addition of di- or trivalent cations within 2 min of the opening procedure. Addition of purified, baculovirus recombinant-expressed VP6 to native and reconstituted cores led to the formation of DLPs or DLP-like particles, which upon transfection into MA104 cells were infectious. The rescued infectivity likely originated in part from unopened and in part from reconstituted cores. Radiolabelled RV (+) ssRNAs could be packaged into reconstituted cores and DLPs, as indicated by resistance to RNase I digestion. The packaging reaction was, however, not RV RNA sequence-specific, since unrelated ssRNAs, such as those transcribed from HIV-2 cDNAs, were also packaged. The kinetics of packaging of homologous and heterologous RNAs were similar, as evidenced by competitive packaging assays. None of the packaged in vitro engineered RNA segments has so far been rescued into infectious virus.
authors.affiliation: Virologie Moléculaire et Structurale, UMR 2472 du CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cédex, France; Molecular Immunology Group, International Centre for Genetic Engineering, Trieste, Italy; Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. Electronic address: ud207@medschl.cam.ac.uk.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Desselberger
authors.forename: James
authors.initials: J
authors.lastname: Richards
authors.forename: Luba
authors.initials: L
authors.lastname: Tchertanov
authors.forename: Jean
authors.initials: J
authors.lastname: Lepault
authors.forename: Andrew
authors.initials: A
authors.lastname: Lever
authors.forename: Oscar
authors.initials: O
authors.lastname: Burrone
authors.forename: Jean
authors.initials: J
authors.lastname: Cohen
mesh_terms: D000818:Animals; D002460:Cell Line; D000882:Haplorhini; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins
keywords: RNA packaging in vitro; Rotavirus; Rotavirus cores